Isolation and characterization of calmodulin and a novel calcium-binding protein calpollenin from Pinus yunnanensis pollen

Abstract

A protein identifiable as calmodulin (CaM) was separated from Pinus yunnanensis pollen by phenyl-sepharose affinity chromatography and further purified by preparative polyacrylamide gel electrophoresis (PAGE). This pollen CaM is heat stable, could activate cAMP phosphodiesterase in a Ca 2+-dependent fashion, and showed the migration change on sodium dodecyl sulfate (SDS)-PAGE in the presence or absence of Ca 2+. Its UV absorption spectrum showed five characteristics peaks. One third of its amino acid composition was acidic amino acids, and the Phe/Tyr ratio was 8:2. In general, the properties of CaM from Pinus yunnanensis pollen is similar to CaMs from animal and other plant sources. A novel 21-kDa calcium-binding protein (CaBP), given the name of calpollenin, was isolated and purified from P. yunnanensis pollen by following the same procedure as for CaM extraction. It was extracted more easily by Triton-containing buffer, and precipitated reversibly by 3.0% (w/v) trichloroacetic acid. Similar to CaM, this calpollenin could bind to a hydrophobic ligand in a Ca 2+-dependent manner, and showed an altered electrophoretic mobility on SDS-PAGE in the presence or absence of Ca 2+. However, calpollenin was heat unstable, and was denatured on heating at 90°C for 5 min. Its UV absorption spectrum showed a single peak at 275 nm. Analysis of amino acid composition demonstrated that calpollenin contained approximately 213 amino acid residues, among which there was a higher proportion of acidic (24.8%) compared with basic amino acids (5.2%).